Voltage dependent Ca2+ channels are transmembrane proteins, which allow Ca2+ entry upon activation. In addition to their electrogenic role, Ca2+ channels provide a pivotal link between membrane depolarization and a wide range of cellular functions. Ca2+ action is often local and close to its source of entrance. Ca2+ action is also very specific. Ca2+ influx through different types of Ca2+ channels can activate distinct cellular signaling cascades. For instance, compared to L-type Ca2+ channels, Ca2+ influx through NMDA receptors activates a distinct signaling pathway for regulation of gene expression. With its abundant and varied intracellular targets, how is Ca2+ able to achieve specificity and activate only a subset those targets in neurons? The long-term goal is to understand the role and molecular mechanisms of Ca2+ channels in neuronal signaling. Specifically, in this proposal, we will test the hypothesis that specific interactions exist between Ca2+ channels and certain other intracellular proteins. Such interactions are of functional significance. Specific aims include isolation and characterization of proteins, which interact with Ca2+ channels. Using yeast two-hybrid system, we have screened a brain cDNA library with the C-termini of three different Ca2+ channel alpha1-subunits as baits. 177 clones have been sequenced. Three clones have been selected for functional studies. They include tctex-1, a light chain of the dynein complex; clones L157 and N397, two distinct forms of PKC binding proteins. Experiments are in progress to address the functional significance of the interactions between Ca2+ channels and those clones in the following aspects: (1) differential distribution of different types of Ca2+ channels in neurons (tctex-1); and (2) modulation of channel activities by PKC and/or initiation of the PKC signaling cascade (L157 & N397). Preliminary data indicate that the interaction between Ca2+ channels and these clones we selected indeed bears the functional significance as we had hypothesized.